Chronic lymphocytic leukemia prognosis and treatment

ABSTRACT

Provided herein are methods for identifying a subject afflicted with chronic lymphocytic leukemia who is responsive to treatment with a chemotherapeutic agent by detecting the presence or absence of at least one APOE4 allele in the subject, the presence of an APOE4 allele identifying the subject as responsive to the treatment. Also provided are methods of treating a subject afflicted with chronic lymphocytic leukemia, including administering an estrogenic agent, an androgen withdrawal agent, an apoE4 peptide or mimetic thereof, and/or a chemotherapeutic agent in an amount effective to treat said chronic lymphocytic leukemia. Methods of determining a prognosis for a patient diagnosed with chronic lymphocytic leukemia are also provided. In addition, methods for stratifying a subject into a subgroup of a clinical trial and methods for identifying a patient in a clinical trial of a treatment for chronic lymphocytic leukemia are herein provided.

RELATED APPLICATIONS

This application is a continuation and claims priority to U.S. patentapplication Ser. No. 13/010,005, filed Jan. 20, 2011, now allowed, whichis a divisional of U.S. patent application Ser. No. 12/265,079, filedNov. 5, 2008, now U.S. Pat. No. 7,902,147, and claims the benefit under35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No.60/985,365, filed Nov. 5, 2007, the disclosure of each of which isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under NIH grant numberCA90548. The Government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns the prognosis and treatment of subjectsafflicted with chronic lymphocytic leukemia.

BACKGROUND OF THE INVENTION

B-cell chronic lymphocytic leukemia (CLL), the most common leukemia inNorth America, is a disease of CD5+ B lymphocytes characterized by slowproliferation and decreased apoptosis. The decreased apoptotic death ofCD5+ B lymphocytes contributes to their increased abundance in blood,producing the clinical entity of CLL.

The progression of this disorder is often indolent, with a mediansurvival over 20 years from the time of diagnosis (Chiorazzi et al.(2005) N Engl J Med 352, 804-15; Keating, M. J. (2002) in Leukemia, eds.Henderson et al. (Saunders, Philadelphia), pp. 131-151). CLL ischaracterized by overexpression of anti-apoptotic proteins such asbcl-2, and commonly employed therapies in CLL increase apoptotic celldeath. Activation of the PI-3-K/Akt pathway in CLL cells can inhibitapoptotic death (Cuni et al. (2004) Leukemia 18, 1391-400).

Many therapies for CLL cause leukemia cell death by triggeringapoptosis. However, there is need for more effective prognosis andtreatment options for diseases such as CLL.

SUMMARY OF THE INVENTION

Provided herein are methods for identifying a subject (e.g., a humansubject) responsive to treatment with a chemotherapeutic agent, whereinthe subject is afflicted with chronic lymphocytic leukemia, includingdetecting the presence or absence of at least one APOE4 allele in abiological sample of the subject, wherein the presence of at least oneAPOE4 allele identifies the subject as a subject whose chroniclymphocytic leukemia is responsive to treatment with thechemotherapeutic agent. In some embodiments, the subject is female. Insome embodiments, the subject is male. In some embodiments, the subjectis an adult or geriatric subject. In some embodiments, the subject is apostmenopausal female subject.

In some embodiments the methods include administering to a subject withat least one APOE4 allele a chemotherapeutic agent (e.g., bendamustine,flavopiridol, fludarabine, chlorambucil, cyclophosphamide, doxorubicin,prednisone, vincristine, monoclonal antibodies such as rituximab,alemtuzumab, lumiliximab, epratuzumab, ofatumumab, etc.) in an amounteffective to treat said chronic lymphocytic leukemia.

Also provided are methods of treating a subject afflicted with chroniclymphocytic leukemia including: (i) detecting the presence or absence ofan APOE4 allele in a biological sample of said subject (e.g., bygenotyping); and (ii) administering (e.g., by oral or parenteraladministration) to said subject an estrogenic agent or androgenwithdrawal agent in an amount effective to treat said chroniclymphocytic leukemia.

Further provided are methods of treating a subject afflicted withchronic lymphocytic leukemia including administering to the subjectapoE4 or mimetic thereof (e.g., by oral or parenteral administration) inan amount effective to treat the chronic lymphocytic leukemia. In someembodiments, the methods include detecting the presence or absence of atleast one APOE4 allele in a biological sample of the subject (e.g., bygenotyping).

Methods of treating a subject afflicted with chronic lymphocyticleukemia are provided, including administering to said subject apoE4 ora mimetic thereof in combination with administering to the subject anestrogenic agent or androgen withdrawal agent, the two combined in anamount effective to treat the chronic lymphocytic leukemia.

Methods of determining a prognosis for a patient diagnosed with chroniclymphocytic leukemia are also provided, including obtaining a patientprofile (e.g., including detecting the presence or absence of at leastone APQE4 allele in a biological sample of the patient, determining thegender of the patient, etc.), and then converting the patient profileinto the prognosis, wherein the presence of said APOE4 allele identifiesthe subject as a subject whose chronic lymphocytic leukemia isresponsive to treatment with one or more chemotherapeutic agents. Insome embodiments, obtaining the patient profile also includes detectingone or more factors selected from the group consisting of: LPL mRNAlevel, clinical stage, lymphocyte doubling time, immunoglobulin IgV_(H)mutation status, cytogenetic abnormalities, leukemia cell CD38expression and Zap-70 expression.

Further provided are methods for stratifying a subject into a subgroupof a clinical trial of a therapy for the treatment of chroniclymphocytic leukemia, including detecting the presence or absence of atleast one APOE4 allele in a biological sample of the subject, whereinthe subject is stratified into said subgroup for said clinical trial ofsaid therapy based upon the presence or absence of said at least oneAPOE4 allele. In some embodiments, the therapy includes administering achemotherapeutic agent.

In some embodiments, the methods include determining the gender of thesubject, wherein the subject is stratified into said subgroup for saidclinical trial of said therapy based upon both: (a) the presence orabsence of said at least one APOE4 allele; and (b) the gender of saidpatient.

Methods are also provided for identifying a patient in a clinical trialof a treatment for chronic lymphocytic leukemia, including: (a)identifying a patient diagnosed with chronic lymphocytic leukemia; and(b) determining a prognosis for the patient diagnosed with chroniclymphocytic leukemia, including obtaining a patient profile (e.g.,including detecting the presence or absence of at least one APOE4 allelein a biological sample of the patient, determining the gender of thepatient, etc.), wherein the prognosis includes a prediction of whetherthe patient is a candidate for the clinical trial. In some embodiments,obtaining the patient profile also includes detecting one or morefactors selected from the group consisting of: LPL mRNA level, clinicalstage, lymphocyte doubling time, immunoglobulin IgV_(H) mutation status,cytogenetic abnormalities, leukemia cell CD38 expression and Zap-70expression.

A further aspect of the present invention is the use of active agents asdescribed herein for the preparation of a medicament for carrying out amethod of treatment for chronic lymphocytic leukemia as describedherein.

The present invention is explained in greater detail in the drawingsherein and in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. APOE4, survival, and time-to-treatment (TTT). 1A. APOE4 andsurvival in females only. 1B. APOE4 and survival in males only. 1C.APOE4 and TTT in the women only. 1D. APOE4 and TTT in men only.

FIG. 2. Frequencies of APOE genotypes and alleles. Numbers above thebars represent numbers of subjects in that particular genotype. Controlvalues are from reference number 27.

FIGS. 3A-3D. Lipoprotein lipase (LPL), survival, and TTT. 3A. LPL andsurvival in women only. 3B. LPL and survival in men only. 3C. LPL andTTT in females. 3D. LPL and TTT in males.

FIGS. 4A-4C. APOE4 genotype/LPL and survival. 4A. APOE4/LPL and survivalin the entire cohort. 4B. APOE4/LPL and survival in females only. 4C.APOE4/LPL and survival in men only. The data are from subjects on whomwe performed both APOE genotyping and LPL mRNA quantitation.

FIG. 5. Proposed model of CLL and the interaction of ApoE4-VLDL and LPLvia cell surface receptors. ApoE4-VLDL tethers to a cell surfacereceptor (either a heparan sulfate proteoglycan or an LDL-receptorfamily member) thereby recruiting SHIP-2, which in turn increases cellapoptosis (4). VLDL particles are converted to LDL particles by thelipase activity of LPL that can also bind to cell surface heparansulfate proteoglycans. CLL patients with an APOE4 genotype (but notAPOE2 or APOE3 genotype) would thus have increased apoptosis of theirleukemia cells induced by ApoE4-VLDL and better prognosis, whilepatients with high LPL levels would have lower ApoE4-VLDL, lessapoptosis of leukemia cells, and worse prognosis.

FIGS. 6A-6D. APOE4, survival, and time-to-treatment (TTT). 6A. APQE4 andsurvival in all subjects (male and female). 6B. APOE4 and survival inall subjects (male and female). 6C. APQE4 and TTT in all subjects (maleand female). 6D. APOE4 and TTT in all subjects (male and female).

FIGS. 7A-7B. Survival and TTT in women and men with CLL. 7A. Survival inthe entire cohort of patients with and without an APOE4 allele. 7B. TTTin the entire cohort of patients with and without an APOE4 allele.

FIG. 8. Cytotoxicity for CLL cells from 3 patients (patients “B,” “C,”and “D”) of VLDL purified from an individual of APOE3 homozygousgenotype (“V3/3”) and an individual of APOE4 homozygous genotype(“V4/4”). The lower dotted line denotes zero cytotoxicity, and the upperdotted line denotes 0.5 fraction of cytotoxicity. A fraction ofcytotoxicity of 1.0 signifies 100% cytotoxicity, and a fraction ofcytotoxicity of 0.5 signifies 50% cytotoxicity. VLDL-apoE4/4 was verycytotoxic for CLL cells (fraction cytotoxicity maximum of 0.65 to 0.75,but VLDL-apoE3/3 displayed minimal cytotoxicity even at the highestconcentrations. At low concentrations, VLDL-apoE3/3 was cytoprotectivefor the CLL cells. This signifies that it enhanced viability of the CLLcells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have discovered a significant association betweenthe APOE4 genotype and responsiveness to treatment and survival amongpatients afflicted with chronic lymphocytic leukemia (CLL). Withoutwishing to be bound by theory, it is hypothesized that the beneficialeffect of an APOE4 genotype is mediated through regulation of leukemiacell apoptosis. Our studies suggest that the APOE4 genotype does notalter susceptibility to developing CLL, but does influence CLL outcomeand response to therapies. The present inventors have also discoveredthat apoE4 (protein) is cytotoxic for CLL cells in vitro, but apoE3 isnot cytotoxic for CLL cells in vitro.

The present inventors have also discovered an unexpected and strikinggender difference in the influence of the APOE4 genotype. Women, but notmen, with an APOE4 genotype had markedly longer survival than non-APOE4patients. This female-specific protective effect may relate to themodulation of CLL cell apoptosis by estrogens.

All United States patent references cited herein are to be incorporatedby reference herein in their entirety to the extent they are consistentwith the disclosure herein.

“Chronic lymphocytic leukemia” or “CLL” is a slow-growing type ofleukemia (blood cancer) in which too many lymphoblasts (immature whiteblood cells) are found in the blood and/or bone marrow. It is a diseaseof CD5+B lymphocytes characterized by slow proliferation and decreasedapoptosis. CLL is currently an incurable disease, and the decision totreat this leukemia is based on a variety of factors including declinein other blood elements (e.g., development of anemia orthrombocytopenia), increasing lymphocyte count rates (e.g., a lymphocytedoubling time of less than 1 year), and systemic side effects such asfever and weight loss (Cheson et al. (1996) Blood 87, 4990-7). Clinicalstage, lymphocyte doubling time, immunoglobulin IgV_(H) mutation status,cytogenetic abnormalities, and leukemia cell CD38 and Zap-70 expressionare significantly associated with survival in CLL (Chiorazzi et al.(2005) N Engl J Med 352, 804-15; Keating, M. J. (2002) in Leukemia, eds.Henderson et al. (Saunders, Philadelphia), pp. 131-151).

“Treating” refers to any type of treatment that imparts a benefit to apatient, e.g., a patient afflicted with a disease (e.g., a leukemia). Asubject is “responsive” to treatment if the treatment results in abenefit to the subject, e.g., increases the rate or probability ofsurvival across a period of time (e.g., 10, 20 or 30 years fromdiagnosis). Treating includes actions taken and actions refrained frombeing taken for the purpose of improving the condition of the patient(e.g., the relief of one or more symptoms), delay in the onset orprogression of the disease, lessening of one or more symptoms associatedwith the disease, etc. In some embodiments, treating CLL includes theadministration of an active agent (e.g., apoE4, estrogen, etc.) to asubject in need thereof. In some embodiments treating CLL furtherincludes the administration of a chemotherapeutic agent. This may beaccomplished by, e.g., the combination therapies described below.

The administration of two or more compounds “in combination” or “inconjunction” means that the two compounds are administered closelyenough in time to have an additive and/or synergistic effect. The twocompounds may be administered simultaneously (concurrently) orsequentially. Simultaneous administration may be carried out by mixingthe compounds prior to administration, or by administering the compoundsat the same point in time but at different anatomic sites or usingdifferent routes of administration.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.“Prodrug” refers to compounds that are rapidly transformed in vivo toyield an active agent, for example, by hydrolysis in blood.

The “time-to-treatment” or “TTT” refers to the length of time betweenthe date of diagnosis and initiation of medical treatment (e.g., byadministering one or more chemotherapeutic agents). Treatment of CLLtypically ranges from periodic observation with treatment of infectious,hemorrhagic, or immunologic complications, to a variety of therapeuticoptions, including chemotherapies such as alkylating agents, purineanalogs, combination chemotherapy and monoclonal antibodies, as well astransplant options. Because this disease is generally not curable,typically occurs in middle-aged and elderly adults, and often progressesslowly, it is most often treated in a conservative fashion.

“Subjects” are, in general, human subjects (including “patients”), orother animal subjects (e.g., laboratory animals), and particularlymammalian subjects such as dogs, cats, rabbits, horses, cattle, sheep,etc. for veterinary purposes. In some embodiments, subjects are female.In other embodiments, subjects are male. Subjects may be of any age,including juvenile, adolescent, teenage, young adult, adult, middle-age,elderly and geriatric subjects. Human subjects may also be of anyethnicity, e.g., Caucasian, African-American, Hispanic, Asian, Indian,etc. In some embodiments, human subjects are at least 16, 18, 25 or 30years of age. In other embodiments, human subjects are at least 35, 40,45 or 50 years of age. In further embodiments, human subjects are atleast 60 or 65 years of age. In some embodiments, human subjects arepostmenopausal female subjects.

“Apolipoprotein E” or “apoE” is a protein that combines with andtransports fats (lipids) in the body. There are at least three isoformsof the APOE gene which encodes the apoE protein: APOE2, APOE3 and APOE4.APOE3 is the most common allele in humans.

The “apoE4” isoform of the lipoprotein apoE has been previouslyimplicated as a predisposing factor in Alzheimer's disease and in thedevelopment of atherosclerosis. ApoE4 has been shown to evoke apoptosisin neuronal cells through the LDL receptor-related protein (LRP) andheterotrimeric GTPases (Hashimoto et al. (2000) J. Neurosci. 20,8401-8409). Antisense oligonucleotides to LRP mRNA and the presence ofLRP-associated protein RAP (receptor associated protein) inhibitapoE4-induced apoptosis (Hashimoto et al. (2000) J. Neurosci. 20,8401-8409).

Blood lipoprotein particles can be either pro- or anti-apoptotic. Highdensity lipoprotein (HDL) particles, interacting through asphingosine-1-phosphate receptor 3 (S1P₃), can inhibit apoptosis byactivating the PI-3-K/Akt pathway (4). Very low density lipoprotein(VLDL) particles containing apolipoprotein E4 (apoE4) (but not the apoE3or apoE2 isoforms) inhibit this PI-3-K/Akt pathway. ApoE4 VLDL cellsurface binding can recruit the phosphoinositol phosphatase SHIP-2 tothe plasma membrane, causing a decrease in PIP3 and subsequent reductionin phosphorylation of Akt. These events can result in increasedapoptosis (DeKroon et al. (2006) Circ Res 99, 829-36). VLDL ismetabolized to low density lipoproteins (LDL) through lipoprotein lipase(LPL).

“Lipoprotein lipase” or “LPL” is a secreted enzyme that hydrolyzesphospholipids in VLDL particles, thereby increasing serum triglyceridesand converting VLDL particles to LDL particles. Higher levels ofleukemia cell LPL mRNA are generally associated with a shorter survivaland shorter TTT in CLL.

“Genotyping” or genotype determination of subjects (i.e., determining ordetecting whether or not a subject carries one, two, or no APOE4alleles) can be carried out in accordance with known techniques, e.g.,as described in U.S. Pat. Nos. 6,027,896 and 5,508,167. Genotypingherein includes “phenotyping” by determining or detecting which apoEprotein is expressed (e.g., by using apoE isoform-specific antibodies,such as those described in U.S. Pat. Nos. 5,767,248 and 5,716,828 toRoses et al.), thereby indicating the presence of one or more APQE4alleles.

“Active agents” of the present invention include any compound thatpromotes the binding of apoE4 to cell surface LDL receptors, or promotesupstream or downstream signaling associated with the presence of theapoE4 protein. See, e.g., U.S. Pat. Nos. 7,220,833 and 7,208,481.Examples of active agents include, but are not limited to, apoE4 (e.g.,VLDL-apoE4) and mimetics thereof.

“Mimetics” of apoE4 are those compounds which are capable of binding LDLreceptors and/or promote upstream or downstream signaling associatedwith the presence of the apoE4 protein. Mimetics may be developed, e.g.,by generating a library of molecules, selecting for those moleculeswhich act as agonists, and identifying and amplifying the selectedagonists. See, e.g., Kohl et al., Science 260, 1934 (1993) (synthesisand screening of tetrapeptides for inhibitors of farnesyl proteintransferase, to inhibit ras oncoprotein dependent cell transformation).Techniques for constructing and screening combinatorial libraries ofoligomeric biomolecules to identify those that specifically bind to agiven receptor protein are known. Suitable oligomers include peptides,oligonucleotides, carbohydrates, nonoligonucleotides (e.g.,phosphorothioate oligonucleotides; see Chem. and Engineering News, page20, Feb. 7, 1994) and nonpeptide polymers (see, e.g., “peptoids” ofSimon et al., Proc. Natl. Acad. Sci. USA 89, 9367 (1992)). See also U.S.Pat. No. 5,270,170 to Schatz; Scott and Smith, Science 249, 386-390(1990); Devlin et al., Science 249, 404406 (1990); Edgington,BIO/Technology 11, 285 (1993). Peptide libraries may be synthesized onsolid supports, or expressed on the surface of bacteriophage viruses(phage display libraries). Known screening methods may be used by thoseskilled in the art to screen combinatorial libraries to identifyantagonists. Techniques are known in the art for screening synthesizedmolecules to select those with the desired activity, and for labelingthe members of the library so that selected active molecules may beidentified. See, e.g., Brenner and Lerner, Proc. Natl, Acad. Sci. USA89, 5381 (1992) (use of genetic tag to label molecules in acombinatorial library); PCT US93/06948 to Berger et al., (use ofrecombinant cell transformed with viral transactivating element toscreen for potential antiviral molecules able to inhibit initiation ofviral transcription); Simon et al., Proc. Natl. Acad. Sci., USA 89, 9367(1992) (generation and screening of “peptoids”, oligomeric N-substitutedglycines, to identify ligands for biological receptors); U.S. Pat. No.5,283,173 to Fields et al. (use of genetically altered Saccharomycescerevisiae to screen peptides for interactions).

In protein or peptide molecules which interact with a receptor, theinteraction between the protein or peptide and the receptor generallytakes place at surface-accessible sites in a stable three-dimensionalmolecule. By arranging the critical binding site residues in anappropriate conformation, mimetics that have the essential surfacefeatures of the peptides described herein may be generated andsynthesized in accordance with known techniques. Methods for determiningpeptide three-dimensional structure and analogs thereto are known, andare sometimes referred to as “rational drug design techniques.” See,e.g., U.S. Pat. No. 4,833,092 to Geysen; U.S. Pat. No. 4,859,765 toNestor; U.S. Pat. No. 4,853,871 to Pantoliano; U.S. Pat. No. 4,863,857to Blalock; (applicants specifically intend that the disclosures of allU.S. patent references cited herein be incorporated by reference hereinin their entirety). See also Waldrop, Science 247, 28029 (1990);Rossmann, Nature 333, 392 (1988); Weis et al., Nature 333, 426 (1988);James et al., Science 260, 1937 (1993) (development of benzodiazepinepeptidomimetic compounds based on the structure and function oftetrapeptide ligands).

In general, those skilled in the art will appreciate that minordeletions or substitutions may be made to the amino acid sequences ofproteins or peptides of the present invention without unduly adverselyaffecting the activity thereof. Thus, peptides containing such deletionsor substitutions are a further aspect of the present invention. Inpeptides containing substitutions or replacements of amino acids, one ormore amino acids of a peptide sequence may be replaced by one or moreother amino acids wherein such replacement does not affect the functionof that sequence. Such changes can be guided by known similaritiesbetween amino acids in physical features such as charge density,hydrophobicity/hydrophilicity, size and configuration, so that aminoacids are substituted with other amino acids having essentially the samefunctional properties. For example: Ala may be replaced with Val or Ser;Val may be replaced with Ala, Leu, Met, or Ile, preferably Ala or Leu;Leu may be replaced with Ala, Val or Ile, preferably Val or Ile; Gly maybe replaced with Pro or Cys, preferably Pro; Pro may be replaced withGly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replacedwith Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replacedwith Pro or Cys, preferably Cys; His may be replaced with Phe or Gln,preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferablyHis or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe orTrp; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may bereplaced with Gln or Ser, preferably Gln; Gln may be replaced with His,Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced withGln, Thr, Pro, Cys or Ala; Thr may be replaced with Gln or Ser,preferably Ser; Lys may be replaced with Gln or Arg; Arg may be replacedwith Lys, Asp or Glu, preferably Lys or Asp; Asp may be replaced withLys, Arg, or Glu, preferably Arg or Glu; and Glu may be replaced withArg or Asp, preferably Asp. Once made, changes can be routinely screenedto determine their effects on function.

“Estrogenic agents” refers to compounds which activate estrogenreceptors and/or are structurally similar to (e.g., are derived from)estrogen (e.g., estrogen receptor agonists). Examples include, but arenot limited to, estrogen and estrogen analogs. See U.S. Pat. No.6,432,643 to Einstein et al.

Without wishing to be bound to a particular theory, the female-specificprotective effective of the APOE4 genotype on survival in CLL couldrelate to several mechanisms, including modulation of CLL cell apoptosisby estrogen through the apoE/Akt pathway. Some studies have indicatedthat estrogens can be cytotoxic for CLL cells in vitro (Huang et al.(2000) Nature 407, 390-5), and diethylstilbestrol treatment of prostatecancer in patients with CLL has been reported to reduce blood CLL cellcounts (Narasimhan et al. (1980) Am J Hematol 8, 369-75). Severalphysiological metabolites of endogenous estrogen (including2-methoxyestradiol) induce apoptosis of leukemia cells by inactivatingAkt (Gao et al. (2005) Oncogene 24, 3797-809). Estrogen receptoragonists stimulate PIP3 synthesis and enhances Akt phosphorylation inendothelial cells (Haynes et al. (2000) Circ Res 87, 677-82). We haveshown that Akt phosphorylation is inhibited by APOE4-VLDL (DeKroon etal. (2006) Circ Res 99, 829-36). Estrogen regulates APOE expression(Wang et al. (2006) Proc Natl Acad Sci USA 103, 16983-8). Androgeninteracts with apolipoprotein genotype, protecting against APOE4-inducedcognitive deficits (Raber et al. (2002) J Neurosci 22, 5204-9). The APOEgenotype alters lipoprotein particle distribution and number, andtriglyceride metabolism, and gender differences in these effects havebeen reported (Dallongeville et al. (1992) J Lipid Res 33, 447-54;Ferrieres et al. (1994) Arterioscler Thromb 14, 1553-60). Theseobservations suggest that the female-specific protective effective ofthe APOE4 genotype on survival in CLL could relate to the modulation ofCLL cell apoptosis by estrogen through the apoE/Akt pathway.

“Estrogen” includes, but is not limited to, naturally occurringestrogens such as estradiol (E₂), estrone (E₁), and estriol (E₃),synthetic conjugated estrogens, oral contraceptives and sulfatedestrogens. See, e.g., Gruber et al. (2002) N Engl J Med 346, 340-352.

“Analogs” of estrogen are compounds that are estrogen-like in that theyhave at least one of the effects of estrogen, e.g., bind to one or moretypes of estrogen receptors in the body (e.g., diethylstilbestrol),and/or are structurally similar to estrogen (e.g., 2-methoxyestradiol).Natural and synthetic estrogens include, but are not limited to,conjugated equine estrogen, ethinyl estradiol, micronized estradiol, 17βestradiol, mestranol, estradiol valerate, 11-nitrato estradiol,7-α-methyl-11-nitrato-estradiol, piperazine estrone sulfate,quinestranol, and 8,9-dehydroestereone (particularly alkali metal saltsand sulfate esters thereof). See, e.g., U.S. Pat. No. 5,422,119 atcolumn 6; U.S. Pat. No. 5,288,717; U.S. Pat. No. 7,138,426 to DiNinno etal.; U.S. Pat. No. RE39,419 to Day et al.

“Estrogen replacement therapy” (sometimes also referred to as “hormonereplacement therapy” or HRT) refers to a long-term therapy in whichestrogen or estrogenic agents are administered to a subject continuouslyover an extended period of time (e.g., one month, one year, or more) tomaintain sustained blood levels of the agent, e.g., to combat theeffects of menopause or hysterectomy which may include the loss ofcalcium from bone and increased incidence of classical osteoporoticfractures of the forearm and hip, ischemic heart disease, etc.Administration may be daily or periodically in some embodiments. See,e.g., U.S. Pat. No. 6,828,103 to Herrington et al.

Administration of estrogen to men and/or the withdrawal of androgen inmen is also contemplated. The withdrawal of androgen in men is known inthe art, for example, in the context of treatment for prostate cancer,by e.g., castration, alterations in gene expression and/or through theuse of an anti-androgenic drug. Accordingly, “androgen withdrawalagents” are administered in one aspect of the present invention.Androgen withdrawal agents, include, but are not limited to, ananti-androgenic drug, including, but not limited to, casodex, nafarelin,leuprolide, goserelin, buserelin, cyproterone acetate, zanoterone,megestrol acetate, hydroxy-progesterone caproate, medrogestone,hydroxyflutamide, Casodex®, nilutamide, finasteride, etc. See, e.g.,U.S. Pat. No. 7,250,180 to Arellano; U.S. Pat. No. 5,723,455 to Tanabeet al.

Active agents may be prepared as a pharmaceutically acceptable salt orester, in accordance with known techniques. Dosage of the active agentwill depend, among other things, the condition of the subject as well asother factors that may be considered to make a prognosis. Optimizationof dosages are within the skill of those in the relevant art.

Combination Therapies.

In some embodiments active agents are administered in conjunction withone or more chemotherapeutic agents and/or radiation therapy and/ortransplantation (e.g., stem cell transplantation), as is known in theart pertaining to the treatment of leukemias.

“Chemotherapeutic” or “antineoplastic” agents as used herein refer toagents in addition to or other than the active agents described above(apoE4 or mimetics thereof, estrogenic agents, androgen withdrawalagents, etc). Chemotherapeutic agents are well known in the art, andinclude, but are not limited to, alkylating agents (e.g., platinumalkylating agents, nitrosaurea alkylating agents, nitrogen mustardalkylating agents, etc.), antimetabolites (e.g., purine, pyrimidine,folic acid, etc.), antibiotic agents (e.g., anthracycline antibiotics,etc.), plant alkaloids (e.g., vinca alkaloids, taxane alkaloids, etc.),topoisomerase inhibitors (e.g., camptotheca, podophyllum, etc.),immunotherapy agents, kinase inhibitors (e.g., cyclin-dependent kinase(cdk) inhibitors such as rocovitine, flavopiridol, etc.), and others(e.g., UCS-01, altretamine, amsacrine, maytansine, etc.). See also U.S.Patent Application Publication No. 2006/0121539 to Debinski et al. atparagraph [0076]; U.S. Patent Application Publication No. 2007/0219268to Hausheer at paragraph [0119].

Examples of alkylating agents include, but are not limited to, BBR3464,bendamustine, busulfan, carboplatin, carmustine, chlorambucil,cisplatin, cyclophosphamide, dacabazine, fotemustine, ifosfamide,lomustine, mechlorethamine hydrochloride, melphalan, oxaliplatin,procarbazine, streptozotocin, temozolomide, thiotepa, uracil mustard,etc. Examples of antimetabolites include, but are not limited to,aminopterin, capecitabine, cladribine, clofarabine, cytarabine,floxuridine, fludarabine phosphate, fluorouracil, gemcitabine,hydroxyurea, mercaptopurine, methotrexate, pemetrexed, raltitrexed,thioguanine, etc. Examples of antibiotic agents include, but are notlimited to, actinomycin-D, bleomycin, daunorubicin, doxorubicin,epirubicin, idarubicin, mitomycin, mitoxantrone, pentostatin,plicamycin, valrubicin, etc. Examples of plant alkaloids include, butare not limited to, docetaxil, paclitaxil, vinblastine, vincristine,vindesine, vinorelbine, etc. Examples of topoisomerase inhibitorsinclude, but are not limited to, camptothecin, etoposide, irinotecan,teniposide, topotecan, etc.

Examples of immunotherapy chemotherapeutic agents include, but are notlimited to, alemtuzumab, bevcizumab, cetuximab, gemtuzumab, ofatumumab,ozogamisin, penitumumab, rituximab, tositumomab, trastuzumab, etc.Monoclonal antibodies directed against the CD20 (rituximab, RIT) andCD52 antigens (campath-1H, alemtuzumab, ALT) have shown beneficialactivity in chronic lymphocytic leukemia (Robak (2004) Leukemia &Lymphoma 45, 205-219). Monoclonal antibodies directed against the CD23antigen (Lumiliximab) and CD22 antigen (Epratuzumab) have also beendeveloped. Other antigens to which antibodies have been prepared (e.g.,for treating B cell leukemias) include, but are not limited to, CD21,CD23, CD22, CD19, CD40, CD37, etc. See, e.g., U.S. Pat. No. 6,896,885 toHanna.

Formulations and Administrations.

The present invention may be carried out in like manner as described inU.S. Pat. No. 6,514,992 to Lee et al. For therapeutic use the activeagents of the present invention will generally be administered in astandard pharmaceutical composition obtained by admixture with apharmaceutical carrier or diluent selected with regard to the intendedroute of administration and standard pharmaceutical practice. Forexample, they may be administered orally in the form of tabletscontaining such excipients as starch or lactose, or in capsule, ovulesor lozenges either alone or in admixture with excipients, or in the formof elixirs or suspensions containing flavoring or coloring agents. Theymay be injected parenterally, for example, intravenously,intramuscularly or subcutaneously. For parenteral administration, theyare best used in the form of a sterile aqueous solution which maycontain other substances, for example, enough salts or glucose to makethe solution isotonic with blood. Tie choice of form for administrationas well as effective dosages will vary depending, inter alia, on thecondition being treated. The choice of mode of administration and dosageis within the skill of the art.

The compounds contemplated for use according to the present invention ortheir pharmaceutically acceptable salts which are active when givenorally, can be formulated as liquids, for example syrups, suspensions oremulsions, tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s) for example, ethanol, glycerin, non-aqueous solvent, forexample polyethylene glycol, oils, or water with a suspending agent,preservative, flavoring or coloring agent.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidformulations. Examples of such carriers include magnesium stearate,starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, pellets containing the activeingredient can be prepared using standard carriers and then filled intoa hard gelatin capsule; alternatively, a dispersion or suspension can beprepared using any suitable pharmaceutical carrier(s), for exampleaqueous gums, celluloses, silicates or oils and the dispersion orsuspension then filled into a soft gelatin capsule. Preferably thecomposition is in unit dose form such as a tablet or capsule.

Typical parenteral compositions consist of a solution or suspension ofthe compound or pharmaceutically acceptable salt in a sterile aqueouscarrier or parenterally acceptable oil, for example polyethylene glycol,polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.Alternatively, the solution can be lyophilized and then reconstitutedwith a suitable solvent just prior to administration.

A typical suppository formulation comprises a compound or apharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent suchas polymeric glycols, gelatins or cocoa butter or other low meltingvegetable or synthetic waxes or fats.

The pharmaceutically acceptable compounds of the invention will normallybe administered to a subject in a daily dosage regimen. For a patientthis may be, for example, from about 0.001 to about 100 mg/kg,preferably from about 0.001 to about 10 mg/kg animal body weight. Adaily dose, for a larger mammal is preferably from about 1 mg to about1000 mg, preferably between 1 mg and 500 mg or a pharmaceuticallyacceptable salt thereof, calculated as the free base, the compound beingadministered 1 to 4 times per day. Unit dosage forms may contain fromabout 25 μg to about 500 mg of the compound.

The active agents may be administered by any medically appropriateprocedure, e.g., normal intravenous or intra-arterial administration.

Active agents may be provided in lyophilized form in a sterile asepticcontainer or may be provided in a pharmaceutical formulation incombination with a pharmaceutically acceptable carrier, such as sterilepyrogen-free water or sterile pyrogen-free physiological salinesolution.

Methods of Determining a Prognosis.

To determine a prognosis (the prospect of recovery as anticipated fromthe usual course of disease or peculiarities of the case) according tosome embodiments of the present invention, diagnostic data, includingthe patient's diagnosis and genetic data, such as the patient's APOEgenotype, may be processed to provide therapeutic options and outcomepredictions. Processing may include obtaining a “patient profile” suchas the collection of a patient's medical history including age andgender, APOE genotyping (e.g., using appropriately designed primers andusing an RT-PCR or PCR amplification step and/or apoE phenotyping, e.g.,using an antibody-mediated method or enzymatic test), and statistical orother analyses that converts this raw data into a prognosis. Theprognosis may include a prediction of a patient's response to drugtherapy, recovery time, time to treatment, treatment efficacy,rehabilitation time, etc. For example, the presence of an APOE4 allelecould be used by those of skill in the art (e.g., treating physicians)as a positive predictor for CLL patients that respond well tochemotherapeutic therapy. In some embodiments, the prognosis may includethe use of a computer software program to analyze patient data and runstatistical cross-checks against relational databases in order toconvert the patient data or profile to a prognosis.

A “patient profile” includes data and/or materials pertaining to thepatient for whom the prognostic analysis is being performed. Data mayinclude information on the patient's diagnosis, age, gender, and/or APOEgenotype. The patient profile may also include materials from thepatient such as blood, serum protein samples, cerebrospinal fluid, orpurified RNA or DNA.

“Factors” that are significantly associated with survival in CLLpatients include, but are not limited to, clinical stage, lymphocytedoubling time, immunoglobulin IgV_(H) mutation status, cytogeneticabnormalities, and leukemia cell CD38 and Zap-70 expression (see, e.g.,Chiorazzi et al. (2005) N Engl J Med 352, 804-15; Keating, M. J. (2002)in Leukemia, eds. Henderson et al. (Saunders, Philadelphia), pp.131-151; Damle et al. (1999) Blood. 94, 1840-1847; Hamblin et al. (1999)Blood. 94, 1848-1854; Dohner et al. (2000) New England Journal ofMedicine 343, 1910-1916; Weinberg et al. (2007) Am J Hematol 82,1063-1070). LPL mRNA levels may also be included as a factor todetermine the prognosis of a patient diagnosed with CLL.

Detecting APOE4 Genotype in Clinical Trials.

In addition to use in the prognosis and/or treatment for CLL, detectionof an APOE4 genotype can be used in conducting a clinical trial in likemanner as other genotype information is used to conduct a clinicaltrial, such as described in, e.g., U.S. Pat. Nos. 6,573,049 6,368,797and 6,291,175.

In some embodiments, such methods advantageously stratify or permit therefinement of the patient population (e.g., by division of thepopulation into one or more subgroups) so that advantages of particulartreatment regimens can be more accurately detected, particularly withrespect to particular sub-populations of patients. In some embodiments,such methods comprise administering a test active agent or therapy to aplurality of subjects (a control or placebo therapy typically beingadministered to a separate but similarly characterized plurality ofsubjects) and detecting the presence or absence of an APOE4 genotype asdescribed above in the plurality of subjects. The APOE4 genotype may bedetected before, after, or concurrently with the step of administeringthe test therapy. The influence of one or more detected APOE4 alleles onthe test therapy can then be determined on any suitable parameter orpotential treatment outcome or consequence, including, but not limitedto, the efficacy of said therapy, lack of side effects of the therapy,etc.

Accordingly, in some embodiments, knowing or detecting APOE genotypes ina randomized research trial will helps investigators balance groups toavoid any bias introduced by certain prognostic factors. For example, inan embodiment comparing treatments X and Y, there should be an equalnumber of APOE4 women in both limbs of the trial. Otherwise, iftreatment X group had more APOE4 women, it would appear that treatment Xwas better than treatment Y, when the results were, in fact, biased bythe number of APOE4 women in the respective groups.

A clinical trial can be set up to test the efficacy of test compounds totreat any number of diseases for which an APOE genotype has beendetermined to be associated with a subject diagnosed with a disease orat risk for developing the disease. If subjects are genotyped after thecompletion of a clinical trial, the analyses may still be aimed atdetermining a relationship between a treatment for a disease and theallele to be assessed for efficacy. Alternatively, if a symptomaticsubject has not yet been diagnosed with the disease but has beendetermined to be at risk, a similar clinical trial to the clinical trialdescribed above may be carried out.

Assessment of the efficacy of a drug chosen for the trial may includemonitoring the subject over a period of time and analyzing the delay ofonset of the disease and the intensity of the disease at the time ofonset, as well as measuring the onset of symptoms which are associatedwith the disease. A drug that, in a clinical trial, eliminates or delaysthe onset of the disease, or reduces the symptoms of the disease may bea beneficial drug to use in patients diagnosed with the disease. Testcompounds which may be used in such trials include chemotherapeuticagents as described above, including those previously approved forclinical use and new compounds not yet approved for use, or approved fortreating CLL in particular. Thus, in some embodiments the clinical trialmay include the optimization of drug administration, including dosage,timing of administration, toxicities or side effects, route ofadministration, and efficacy of the treatment.

The present invention is explained in greater detail in the followingnon-limiting Examples.

Example 1

A cohort of 183 CLL patients (50 females and 133 males) were followed inthe Durham V.A. and Duke University Medical Centers (Table 1). Analysisof this cohort by gender revealed a profound survival benefit for femalepatients with an APOE4 genotype (i.e., possessing either one or twoAPOE4 alleles) (FIG. 1A), but there was no significant benefit for males(FIG. 1B). For the overall cohort (females and males together), APOE4patients had longer survival than those with no APOE4 alleles (i.e., anon-APOE4 genotype) (FIG. 6A), but this difference for the populationwas not statistically significant. No statistically significantdifferences in survival were observed in any of these populationsbetween APOE2 genotype and non-APOE2 genotype patients, or between APOE3genotype and non-APOE3 genotype patients (data not shown). The longsurvival in APOE4 CLL patients is contrary to that of the generalpopulation, in which APOE4 genotype increases the risk foratherosclerosis and death from vascular disease (Davignon et al. (1999)Clin Chim Acta 286, 115-43).

CLL is an incurable disease, and the decision to treat this leukemia isbased on a variety of factors, including decline in other blood elements(development of anemia or thrombocytopenia), increasing lymphocyte countrates (e.g., a lymphocyte doubling time of less than 1 year), andsystemic side effects such as fever and weight loss (Cheson et al.(1996) Blood 87, 4990-7). The time-to-treatment (TTT) from diagnosistherefore reflects the rate of disease progression. The presence orabsence of an APOE4 allele did not alter TTT in females (FIG. 1C) or inmales (FIG. 1D) separately or in the combined cohort of males andfemales (FIG. 6B). Of the APOE4 women, 7 of 12 received chemotherapy andnone of 12 died. Of the non-APOE4 women, 22 of 38 received chemotherapyand 9 of 38 died.

The APOE genotype distribution of the CLL patient population did notdiffer from that of the general population (Schaefer et al. (1994)Arterioscler Thromb 14, 1105-13) (FIG. 2). These data suggest that,although APOE genotype influences survival with CLL, it does notinfluence the risk of developing this disease.

Lipoprotein lipase is a secreted enzyme that hydrolyzes phospholipids inVLDL particles, thereby increasing serum triglycerides and convertingVLDL particles to LDL particles. In agreement with earlier publications(Oppezzo et al. (2005) Blood 106, 650-7; Nuckel et al. (2006) LeukLymphoma 47, 1053-61; Heintel et al. (2005) Leukemia 19, 1216-23),higher levels of leukemia cell LPL mRNA were significantly associatedwith a shorter survival and shorter TTT in the analysis of males andfemales in our CLL cohort independent of APOE status (FIG. 3A-D), butthis was statistically significant only in males. Likewise, in analyzingthe entire cohort of males and females together, those with high LPLmRNA levels had a significantly worse survival and TTT (FIGS. 6C and6D).

We next analyzed survival of patients with CLL according to both APOEgenotype and CLL cell LPL mRNA level (FIG. 4A-C). This co-analysis waspossible in 110 of the 183 patients for whom enough mRNA was availablefor LPL analysis. APOE4 genotype Patients with either low LPL or highLPL had much better survival (100%) than the non-APOE4 patients witheither low LPL or high LPL. This was apparent when the population wasconsidered as a whole (FIG. 4A), for females alone (FIG. 4B), or formales alone (FIG. 4C). These differences were statistically significantfor the group as a whole and for men alone. These results suggest thatAPOE4 genotype has a stronger effect on survival than does LPL level.

TABLE 1 Patient characteristics-Duke University and V.A. Medical CentersDuke & V.A. Duke only V.A. only Number All 183 All 125 All 58 (percent)M 133 (73%) ^(a) M 77 (62%) M 56 (97%) F 50 (27%) F 48 (38%) F 2 (3%)Age at diagnosis All 59 [52-66] ^(b) All 59 [52-66] All 59 [52-64][25^(th)-75^(th) percentile] M 60 [52-65] M 60 [51-66] M 59.5 [54-65] F59 [52-68] F 59 [52-68] F 59 [43-75] Rai stage at diagnosis All116-34-21-4-8 (63%-19-11-2-4) ^(c) All 83-22-14-4-2 ( 66%-18-11-3-2 )^(d) All 33-12-7-0-6 ( 57%-21-13-0-10 ) ^(d) Stages 0-1-2-3-4 M80-27-17-1-8 ( 60%-20-13-1-6 ) ^(e) M 52-18-11-1-2 (62%-21-13-1-2) M33-12-7-0-6 (57%-21-12-0-10) Number F 36-7-4-3-4 ( 72%-14-8-6-8 ) ^(e) F36-8-4-3-1 (69%-15-8-6-2) F 1-1-0-0-0 (50%-50-0-0-0) (Percent) Length offollow-up All 6.1 [3.2-10.8] All 6.3 [3.4-10.8] All 5.5 [2.6-10.8] Years[25^(th)-75^(th) M 5.7 [3.0-10.2] M 6.2 [3.3-9.5] M 5.7 [2.7-10.8]percentile] F 7.2 [3.5-11.9] F 7.2 [3.4-12.0] F 6.4 [2.7-10.0] Treated &Untreated All 102 & 81 ^(a) (56% & 44) All 69 & 56 (55% & 44) All 33 &25 (57% & 43) M 73 & 60 (55% & 45) M 45 & 39 (54% & 46) M 33 & 25 (57% &43) F 29 & 21 (58% & 42) F 30 & 22 (58% & 42) F 1 & 1 (50% & 50) Died &Lived All 34 & 149 ^(a) (19% & 81) All 19 & 106 (15% & 85) All 15 & 43(26% & 74) M 25 & 108 (19% & 81) M 14 & 70 (17% & 83) M 16 & 42 (28% &72) F 9 & 41 (18% & 82) F 11 & 41 (21% & 79) F 0 & 2 (0% & 100%) APOEallele frequency All 8, 77, 15 (366 alleles) All 8, 76, 16 (250 alleles)All 8, 79, 12 (116 alleles) (percent) M 8, 76, 16 (264 alleles) M 8, 73,19 (154 alleles) M 8, 80, 13 (112 alleles) (E2, E3, E4) F 8, 80, 12 (100alleles) F 9, 80, 13 (96 alleles) F 25, 75, 0 (4 alleles) LPL mRNA(units) All 3.36 [0.07-29.5] ^(b) All 1.35 [0.07-21.7] All 8.88[0.11-34.4 (n = 85) (n = 60) (n = 25) M 4.81 [0.06-36.4] M 2.57[0.15-18.48] M 10.95 [0.29-46.26] (n = 69) (n = 45) (n = 24) F 0.98[0.06-36.4] F 1.15 [0.07-37.25] F Not done (n = 16) (n = 15) ^(a) Number(percent) ^(b) Median [25^(th)-75^(th) percentile] ^(c) Stages 0, 1, 2,3, and 4 (percent at each stage) ^(d) p = 0.04. Bold and underlinedregions display the only values that are statistically significantlydifferent (Chi square test) ^(e) p = 0.02 (Chi square test)

Clinical stage, lymphocyte doubling time, immunoglobulin IgV_(H)mutation status, cytogenetic abnormalities, and leukemia cell CD38 andZap-70 expression are significantly associated with survival in CLL.Women with CLL and an APOE4 genotype compared to the non-APOE4 womenpatients had a significantly lower Rai stage, were less likely to haveZap-70 positive cells, had a higher hematocrit at the time of diagnosis,and had fewer deaths (Table 2). There were no differences in CD38expression, IgV_(H) mutational status, and cytogenetic abnormalitiesbetween women with and without the APOE4 genotype. High levels of LPLmRNA were significantly associated with unmutated IgV_(H) status, CD38positivity, Zap-70 positivity, advanced clinical stage, high serum LDH,and higher (worse) scores on a CLL prognostic index scale (Weinberg etal. (2007) Am J Hematol 82, 1063-1070) (Table 2).

TABLE 2 Patient characteristics - APOE4 and non-APOE4 All APOE4Non-APOE4 Number All 183 (100%) ^(a) All 53 (100%) All 130 (100%) M 133(73%) M 41 (77%) M 92 (71%) F 50 (27%) F 12 (23%) F 38 (29%) Rai stageat diagnosis All 116-34-21-4-8 (63%-19-11-2-4) ^(b) All 33-10-9-0-1(62%-19-17-0-2) All 83-24-12-4-7 (64%-18-9-3-5) Stages 0-1-2-3-4 M80-27-17-1-8 ( 60%-20-13-1-6 ) ^(c) M 24-9-7-0-1 (59%-22-17-0-2) M56-18-10-1-7 (61%-20-11-1-8) Number F 36-7-4-3-4 ( 72%-14-8-6-8 ) ^(c) F9-1-2-0-0 (75%-8-17-0-0) F 27-6-2-3-0 (71%-16-5-8-0) (Percent) LeukocyteDT All 3.4 All 3.5 All 3.2 (years) M 3.5 [1.3-13.3] ^(d) M 3.3[1.4-12.7] M 3.6 [1.3-13.3] F 2.9 [1.3-13.7] F 4.2 [1.4-13.7] F 2.1[1.1-13.7] IgV_(H) Mutated & All 112 & 63 (64% & 36) All 33 & 16 (67% &33) All 79 & 47 (63% & 37) Unmutated M 82 & 44 (65 & 35) M 27 & 11 (71%& 29) M 55 & 33 (63% & 38) F 30 & 19 (61% & 39) F 6 & 5 (55% & 45) F 24& 14 (63% & 37) CD38 positive & All 51 & 127 (29% & 71) All 11 & 40 (22%& 78) All 40-87 (32% & 69) CD38 negative M 39 & 90 (30% & 70) M 10 & 29(26% & 74) M 29 & 61 (32% & 68) F 12 & 37 (24% & 76) F 1 & 11 (8% & 2%)F 11 & 26 (30% & 0) Zap-70 positive & All 129 & 43 (75% & 25) All 33 &14 (70% & 30) All 96 & 29 (77% & 23) Zap-70 negative M 97 & 28 (78% &22) M 28 & 7 (80% & 20) M 69 & 21 (77% & 23) F 32 & 15 (68% & 32) F5 & 7 ( 42% & 58 ) ^(e) F 27 & 8 ( 77% & 23 ) ^(e) Cytogenetics All 77 &14 (85% & 15) All 19 & 4 (83% & 17) All 58 & 10 (95% & 15) Good & Bad M56 & 8 (88% & 13) M 13 & 2 (87% & 13) M 43 & 6 (88% & 12) F 21 & 6 (78%& 21) F 6 & 2 (75% & 25) F 15 & 4 (79% & 21) Initial WBC (×10⁹/L) All 22[18.4-31] All 20 [16-27] All 24 [19-33] M 22 [18.6-32.5] M 20 [15-29] M24.5 [19-33.25] F 21.5 [18.3-29.5] F 20 [18.75-23.5] F 23 [18.1-31.75]Initial hematocrit All 42 [39-45] All 43 [39-45] All 42 [440-46] (L/L ×100) M 43 [40-46] M 43 [41-47] M 43 [39-44] F 41 [38-44] F 45 [ 43-46 ]^(f) F 39 [ 37-43 ] ^(f) Initial platelet count All 191 [152-243] All183 [129-226] All 200 [163-249] (×10⁹/L) M 185 [147-232] M 183 [132-230]M 190 [160-244] F 211 [173-255] F 190 [124-214] F 134 [185-256] Treated& Untreated All 102 & 81 (56% & 44) All 30 & 23 (57% & 43) All 72 & 58(55% & 45) M 73 & 60 (55% & 45) M 23 & 18 (56% & 44) M 50 & 42 (54% &46) F 29 & 21 (58% & 42) F 7 & 5 (58% & 42) F 22 & 16 (58% & 42) Died &Lived All 34 & 149 (19% & 81) All 6 & 47 (11% & 89) All 28 & 102 (22% &78) M 25 & 108 (19% & 81) M 6 & 35 (15% & 85) M 19 & 73 (21% & 79) F 9 &41 (18% & 82) F 0 & 12 ( 0% & 100 ) ^(g) F 9 & 29 ( 24% & 76 ) ^(g) LPLmRNA (units) All 3.64 [0.07-30.00] All 0.58 [0.05-11.70] All 5.00[0.15-43.33] n = 110 n = 31 ^(h) n = 79 ^(h) M 5.00 [0.15-30.00] M 1.33[0.05-15.65] M 9.40 [0.15-43.33] n = 73 n = 21 ^(i) n = 51 ^(i) F 0.98[0.06-36.38] F 0.07 [0.04-2.14] F 3.56 [0.11-47.85] n = 38 n = 10 ^(j) n= 28 ^(j) ^(a) Number (percent) ^(b) Stages 0, 1, 2, 3, and 4 (percentat each stage) ^(c) p = 0.02. Bold and underlined regions display theonly values that are statistically significantly different (Chi squaretest) ^(d) Median [25^(th)-75^(th) percentile] ^(e) p = 0.03 (Chi squaretest) ^(f) p = 0.009 (Wilcoxon test) ^(g) p = 0.02 (Chi square test)^(h) p = 0.054 (Wilcoxon test) ^(i) p = 0.31 (Wilcoxon test) ^(j) p =0.057 (Wilcoxon test)

High LPL mRNA levels were also significantly associated with thecytogenetic abnormalities 17p13 del, trisomy 12, and 11q22 del, whilelow levels of LPL mRNA were significantly associated with the 13q14 delabnormality. APOE4 individuals tended to have lower LPL levels thannon-APOE4 individuals, but the differences were not statisticallysignificant (Table 3).

TABLE 3 Lipoprotein lipase correlations Number LPL mRNA ¹ p value ²IgV_(H) mutation status vs LPL Mutated 67 0.46 (0.02-7.3) mRNA Unmutated42 18.1 (3.1-56.5) <0.0001 CD38 vs LPL mRNA Positive 23 30.9 (0.8-65.6)Negative 86  1.2 (0.1-14.2) 0.002 Zap-70 ³ vs LPL mRNA Positive 69 12.6(0.4-48.1) Negative 38  0.4 (0.1-6.5) 0.0006 Serum LDH vs LPL mRNA High27 13.6 (1.0-61.6) Normal 75  1.2 (0.1-16.0) 0.009 Cytogenetics vs LPLmRNA Good ⁴ 43  0.3 (0.02-9.4) Bad 8 15.1 (11.9-60.2)  0.004 13q14 del24  0.1 (0.04-1.2) Not 13q14 del 27  9.4 (0.1-30.9) 0.04 17p13 del 454.6 (19.6-101.0)  Not 17p13 del 47 0.4 (0.04-12.5) 0.02 Trisomy 12 629.2 (7.1-109.3)  Not Trisomy 12 45 0.4 (0.03-12.5) 0.02 CLL prognosisscore ⁵ 0 44  0.5 (0.03-7.7) 1 29  2.6 (0.3-22.5) 2 22 41.7 (0.5-71.3) 35 50.5 (6.8-59.6) 0.003 ¹ Units (see methods section) ² Wilcoxon test ³Zap-70 determined by immunoblot (8) ⁴ Cytogenetic abnormalitiesdetermined by FISH analysis. “Good” included 13q14 del and normal. “Bad”included 17p13 del, 11q22 del, trisomy 12, and complex abnormalities. ⁵CLL prognosis score derived from clinical stage, CD38 expression, andserum LDH (8).

TABLE 4 Additional Patient Characteristics - Race, Drug Rx, Hormonal RxRace African American Caucasian Hispanic Indian Unknown Total All 1910.4% 160 87.9% 1 0.5% 1 0.5% 1 0.5% 182 Men 11 8.3% 118 89.4% 1 0.8% 10.8% 1 0.8% 132 Women 8 16.0% 42 84.0% 0 0.0% 0 0.0% 0 0.0% 50 E4 4 7.7%47 90.4% 0 0.0% 1 1.9% 0 0.0% 52 non E4 15 11.5% 113 86.9% 1 0.8% 0 0.0%1 0.8% 130 E4 1 8.3% 11 91.7% 0 0.0% 0 0.0% 0 0.0% 12 Women non E4 718.4% 31 81.6% 0 0.0% 0 0.0% 0 0.0% 38 women E4 men 3 7.5% 36 90.0% 00.0% 1 2.5% 0 0.0% 40 non E4 8 8.7% 82 89.1% 1 1.1% 0 0.0% 1 1.1% 92 menDrug Rx E4 non E4 E4 women non E4 women Fludarabine 11 44 2 12 21.2%33.8% 16.7% 31.6% Cyclophosphamide  6 23 2  5 11.5% 17.7% 16.7% 13.2%Rituximab 10 33 2 10 19.2% 25.4% 16.7% 26.3% Alemtuzumab  2  6 0  2 3.8%4.6% 0.0% 5.3% Chlorambucil 20 48 5 17 38.5% 36.9% 41.7% 44.7% No Rx 3072 5 16 57.7% 55.4% 41.7% 42.1% Total 52 130  12  38 Hormonal Rx E4women non E4 women Climara 0 1 0.0% 0.8% Est patch/Provera 0 3 0.0% 2.3%Est vaginal 1 0 1.9% 0.0% Est/Provera 1 0 1.9% 0.0% Estrogen 0 2 0.0%1.5% Estrogen patch 0 1 0.0% 0.8% Estrogen vaginal 0 3 0.0% 2.3%Premarin 1 5 1.9% 3.8% Premarin/Provera 2 3 3.8% 2.3% Any Hormonal Rx 518  9.6% 13.8%  Total 52  130 

Discussion.

We observe here that the APOE4 genotype is associated with markedlyincreased survival in female CLL patients, but not with the length oftime before treatment is required (TTT). These observations suggest thatthe APOE4 genotype enhances responses to therapy. Therapies commonlyemployed in CLL trigger apoptosis of these cells (Keating, M. J. (2002)in Leukemia, eds. Henderson et al. (Saunders, Philadelphia), pp.131-151; Schwarz et al. (2001) in The chemotherapy source book, ed.Perry, M. C. (Lippincott Williams & Wilkins, Philadelphia), pp. 1-6).Since apoE4-VLDL increases the apoptosis of endothelial cells initiatedby withdrawing growth factors (DeKroon et al. (2006) Circ Res 99,829-36), it may similarly increase apoptosis of CLL cells exposed totherapeutic drugs.

Without wishing to be bound to any particular theory, it is thought thatthe APOE4 allele specificity reported here results from apoEisoform-selective binding of VLDL to cell surface receptors. Inendothelial cells, apoE4-VLDL is pro-apoptotic through its interactionwith an as yet unidentified cell surface receptor that is inhibited bythe receptor-associated protein (RAP). RAP inhibits binding of apoE bothto LDL-family receptor members and to heparan sulfate proteoglycans onthe cell surface (DeKroon et al. (2003) J Lipid Res 44, 1566-73; Ji etal. (1998) J Biol Chem 273, 13452-60). ApoE4-VLDL binding to CLL cellsmay enhance leukemia cell apoptosis initiated by various means (e.g.,chemotherapy) and improve overall survival.

Elevated LPL mRNA or LPL protein levels are associated with poorerprognosis in CLL (Oppezzo et al. (2005) Blood 106, 650-7; Nuckel et al,(2006) Leuk Lymphoma 47, 1053-61; Heintel et al. (2005) Leukemia 19,1216-23). LPL is a secreted lipase that binds heparan sulfateproteoglycans on the cell surface. Furthermore, LPL binds to VLDLparticles (that also bind cell surface heparan sulfate proteoglycans)and hydrolyzes VLDL triglycerides (FIG. 5) (Mulder, M., Lombardi, P.,Jansen, H., van Berkel, T. J., Frants, R. R. & Havekes, L. M. (1993) JBiol Chem 268, 9369-75). LPL thereby converts VLDL particles to LDLparticles and increases serum fatty acids. Elevated LPL enzymaticactivity would therefore increase the rate of conversion of VLDL to LDL.The deleterious effects of elevated LPL activity on survival in CLL maybe due to the reduced abundance of apoE4-VLDL particles, a conditionthat would lead to decreased apoptosis.

Several groups have reported that women have a slightly longer survivalthan men in CLL (see Molica, S. (2006) Leuk Lymphoma 47, 1477-1480 forreview), but this was not the case in our cohort (FIG. 7). We did note,however, that the effect of the APOE4 allele on survival is much morepronounced in women, while the effect of LPL levels on survival is morepronounced in men. Women with CLL generally present with fewerunfavorable clinical features, and respond better to treatment (Molica,S. (2006) Leuk Lymphoma 47, 1477-1480).

The female-specific protective effective of the APOE4 genotype onsurvival in CLL could relate to several mechanisms, including modulationof CLL cell apoptosis by estrogen through the apoE/Akt pathway. In ourcohort, 92% of the women with CLL and an APOE4 genotype werepostmenopausal at the time of diagnosis, but most were taking hormonalreplacement therapy. Of the 50 women (range of age at diagnosis 42-85yr), 45 were postmenopausal, and 23 of 25 (on whom information wasavailable) were receiving hormonal replacement therapy. Estrogens can becytotoxic for CLL cells in vitro (Huang et al. (2000) Nature 407,390-5), and diethylstilbestrol treatment of prostate cancer in patientswith CLL has been reported to reduce blood CLL cell counts (Narasimhanet al. (1980) Am J Hematol 8, 369-75). Several physiological metabolitesof endogenous estrogen (including 2-methoxyestradiol) induce apoptosisof leukemia cells by inactivating Akt (Gao et al. (2005) Oncogene 24,3797-809). Estrogen receptor agonists stimulate PIP3 synthesis andenhances Akt phosphorylation in endothelial cells (Haynes et al. (2000)Circ Res 87, 677-82). We have shown that Akt phosphorylation isinhibited by APOE4-VLDL (DeKroon et al. (2006) Circ Res 99, 829-36).Estrogen regulates APOE expression (Wang et al. (2006) Proc Natl AcadSci USA 103, 16983-8). Androgen interacts with apolipoprotein genotype,protecting against APOE4-induced cognitive deficits (Raber et at (2002)J Neurosci 22, 5204-9). The APOE genotype alters lipoprotein particledistribution and number, and triglyceride metabolism, and genderdifferences in these effects have been reported (Dallongeville et al.(1992) J Lipid Res 33, 447-5426; Ferrieres et al. (1994) ArteriosclerThromb 14, 1553-60). These observations point to a female-specificprotective effective of the APOE4 genotype on survival in CLL thatrelates to the modulation of CLL cell apoptosis by estrogen through theapoE/Akt pathway.

APOE genotyping of patients with CLL provides important clinicalprognostic information. The allele-specific influence of APOE on diseaseprogression also provides important new insights into the mechanisms ofdisease and response to therapy.

The frequency of the APOE alleles in the CLL patient population wasidentical to that of control populations. APOE genotype therefore doesnot appear to affect susceptibility to CLL, but influences the clinicalcourse of disease, particularly after therapy is initiated. In contrast,APOE genotype does influence susceptibility to other diseases, mostnotably Alzheimer's, in which APOE4 markedly increases risk (Saunders etal. (1993) Neurology 43, 1467-72).

Materials and Methods.

Patients with CLL were recruited from the Duke University and V.A.Medical Centers from July 1999 through August 2006 as previouslydescribed (Weinberg et al. (2007) Am J Hematol 82, 1063-1070). The 183patients studied here [from the cohort of 190 patients we reportedearlier (Weinberg et al. (2007) Am J Hematol 82, 1063-1070)] were thosefrom whom we had sufficient DNA to genotype for APOE. Fifty-eightpatients (32%) were from the Durham V.A. Medical Center, and 125patients (68%) were from Duke University Medical Center. One hundredthirty-three patients (73%) were male. Diagnosis and staging of CLL, anddecisions regarding initiation of treatment were determined according toNCI Working Group criteria (Cheson et al. (1996) Blood 87, 4990-7;Weinberg et al. (2007) Am J Hematol 82, 1063-1070). The length of timefrom diagnosis to death from any cause was defined as overall survival,and the length of time to initiation of treatment from the date ofdiagnosis was defined as the time-to-treatment. All subjects had notreceived CLL therapy for at least 4 weeks before blood was sampled, andall patients gave informed consent according to protocols approved bythe V.A. and Duke University Institutional Review Boards.

Blood anticoagulated with sodium heparin was processed to enrich the CLLcells by negative selection using monoclonal antibodies as previouslydescribed (Volkheimer et al. (2007) Blood 109, 1559-67; Weinberg et al.(2007) Am J Hematol 82, 1063-1070). The enriched CLL cells contained0.9±0.1% (mean±SEM) T cells and 3.4±0.6% CD19⁺/CD5⁻ cells (“normal” Bcells). Purified cells were immunophenotyped on the day of isolation,and some were frozen for later analyses. Doubling times, CLL cellphenotypes including CD38 and Zap-70 assessment, IgV_(H) mutationstatus, and FISH analyses were performed as we have previously described(Volkheimer et al. (2007) Blood 109, 1559-67; Weinberg et al. (2007) AmJ Hematol 82, 1063-1070).

APOE genotyping was performed as previously described (Saunders et al.(1993) Neurology 43, 1467-72). We assayed LPL mRNA by quantitativeRT-PCR using TaqMan pre-made primers (Applied Biosystems) forlipoprotein lipase (LPL) and β-actin (ACAB) genes. cDNA was synthesizedwith the high capacity archive kit (Applied Biosystems) using a minimumof 50 ng of RNA. cDNA was amplified using the TaqMan Universal 2×PCR mix(Applied Biosystems). Standard semi-logarithmic curves for each LPL andACAB determination, correlating RNA concentration and Ct values, wereconstructed for each experiment using a standard preparation of highlypurified RNA obtained from pheresis-isolated lymphocytes (Mihovilovic etal. (1993) Methods Neurosc 12, 169-190). LPL values were normalizedusing its corresponding ACAB value. All samples were determined induplicate. We dichotomized LPL values withreceiver-operator-characteristics (ROC) curve analysis using “good” and“poor” prognosis groups. “Good” prognosis was defined as not requiringtreatment for >5.5 years from diagnosis (the 75^(th) percentile fortime-to-treatment), and “bad” prognosis was defined as requiringtreatment within <2.6 years from diagnosis (the 50^(th) percentile fortime-to-treatment). The ROC-determined LPL cutoff value was 6.2 units,with an area under the curve of 0.73. Comparisons of clinical andlaboratory parameters between groups were done using the Wilcoxon's testand the chi square test as appropriate. Survival and time-to-treatmentKaplan-Meier data were analyzed using the log rank test. A two-sidedalpha of 0.05 was used for all tests.

Example 2

We determined if estrogen receptor agonists [2-methoxyestradiol (2-ME)and diethylstilbesterol (DES)] and mixed agonist-antagonist [tamoxifen(TAM)] could kill CLL cells in vitro. Freshly isolated CLL cells wereincubated 72 hours with 2-ME, DES, and TAM at various concentrations.Then the cytotoxicity of the agents was determined.

All were cytotoxic for the CLL cells. The effective dose for 50% killing(ED50) for each was 7.5 μM (2-ME), 8.2 μM (DES), and 6.5 μM (TAM). At 10μM, the percent cytotoxicity for each was 71% (2-ME), 79% (DES), and 99%(TAM).

Example 3

Experiments were performed to determine if apoE4 and apoE3 isolated fromplasma of humans who were homozygous for APOE4 or APOE3 were cytotoxicfor freshly purified CLL cells from patients with CLL (FIG. 8). VLDL waspurified by centrifugation of plasma (over potassium bromide gradients)from individuals who were either homozygous APOE4 (“4/4”) or APOE3(“3/3”). VLDL was measured as μg/ml cholesterol. Cells were purifiedusing “Rosette-Sep” for B cells (Stem Cell Technologies, Vancouver,British Columbia), and were >97% pure CLL cells. Cytotoxicity wasdetermined using the MTS colorimetric assay (Promega, Madison, Wis.)after 72 hours' culture in vitro.

Results showed that very low density lipoprotein (VLDL)-apoE4 wasdirectly cytotoxic for CLL cells in vitro. VLDL is the major source ofplasma apoE4. However, VLDL-apoE3 was only minimally cytotoxic for CLLcells, and at low concentrations of VLDL-apoE3, it was protective forCLL cells (it enhanced cell viability). In parallel experiments, it wasdemonstrated that CLL cells incubated with VLDL-apoE4 had diminishedlevels of phosphorylated Akt, compared to control cells or those treatedwith VLDL-apoE3. While not wishing to be bound by theory, it isgenerally thought that phosphorylated Akt is anti-apoptotic andlife-promoting for CLL cells, and that dephosphorylated Akt enhancesapoptosis and death of CLL cells.

These results indicate that VLDL-apoE4 or mimetics of VLDL-apoE4 may beuseful as a cytotoxic treatment for CLL. VLDL-apoE4 (or a mimetic) couldbe administered as a purified agent orally or parenterally. Also, agentsthat would increase concentrations of VLDL-apoE4 should be cytotoxic forCLL cells. Alternatively, antagonists of VLDL-apoE3 or agents thatreduce concentrations of VLDL-apoE3 should reduce the viability andnumbers of CLL cells.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of treating a subject afflictedwith chronic lymphocytic leukemia comprising administering to saidsubject apoE4 or a mimetic thereof in an amount effective to treat saidchronic lymphocytic leukemia.
 2. The method of claim 1, wherein saidadministering step is carried out by administering to said subjectVLDL-apoE4.
 3. The method of claim 1, further comprising the step ofdetecting the presence or absence of an APOE4 allele in a biologicalsample of said subject.
 4. The method of claim 3, wherein said detectingstep is carried out by genotyping.
 5. The method of claim 1, whereinsaid administering step is carried out by oral administration.
 6. Themethod of claim 1, wherein said administering step is carried out byparenteral administration.
 7. A method of treating a subject afflictedwith chronic lymphocytic leukemia comprising administering to saidsubject apoE4 or a mimetic thereof in combination with administering tosaid subject an estrogenic agent or androgen withdrawal agent, the twocombined in an amount effective to treat said chronic lymphocyticleukemia.
 8. The method of claim 7, wherein said administration step iscarried out by administering to said subject VLDL-apoE4 in combinationwith administering to said subject an estrogenic agent or androgenwithdrawal agent.